2.1. Recombinant DNA Technology and Gene Expression
Recombinant DNA technology involves insertion of specific DNA sequences into a DNA vector (vehicle) to form a recombinant DNA molecule which is capable of replication in a host cell. Generally, the inserted DNA sequence is foreign to the recipient DNA vehicle, i.e., the inserted DNA sequence and the DNA vector are derived from organisms which do not exchange genetic information in nature, or the inserted DNA sequence may be wholly or partially synthetically made. In recent years several general methods have been developed which enable construction of recombinant DNA molecules. For example, U.S. Pat. No. 4,237,224 to Cohen and Boyer describes production of such recombinant plasmids using restriction enzymes and a method known as ligation. These recombinant plasmids are then introduced, by means of transformation, and replicated in unicellular organisms. Another method for introducing recombinant DNA molecules into unicellular organisms is transduction or transfection which utilizes bacteriophage vectors and an in vitro packaging system (see Collins and Hohn in U.S. Pat. No. 4,304,863).
Regardless of the method used for construction, the recombinant DNA molecule must be able to survive and replicate in the host cell. The recombinant DNA molecule should also have a marker function which allows the selection of host cells so transformed (or transduced) by the recombinant DNA molecule. In addition, if all of the proper replication, transcription and translation signals are correctly arranged on the plasmid, the foreign gene will be properly expressed in the transformed cells and their progeny.
The processes of transcription and translation represent two levels of control of gene expression. Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes transcription of a gene or a group of linked genes (operon). Promoters vary in their "strength", i.e., their ability to promote transcription. For the purpose of molecular cloning it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in an E. coli host cell system, any of the promoters isolated from E. coli, its bacteriophages or plasmids may be used. More specifically, the P.sub.R and P.sub.L promoters of coliphage .lambda. direct high levels of transcription of adjacent DNA segments. In addition, the recA and lac promoters from E. coli provide high levels of gene transcription of adjacent fragments. Furthermore, other E. coli promoters or synthetic DNA sequences may be used to provide the signal for transcription of the inserted gene.
Specific initiation signals are also required for efficient translation of messenger RNA (mRNA) in procaryotic cells; such procaryotic signals differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a multipartite ribosome binding site on the mRNA including a short Shine-Dalgarno (SD) nucleotide sequence and the start codon (AUG) (located just 3' to the SD sequence) which codes for the amino-terminal methionine of the protein. The SD sequences have complementarity to the 3'-end of the 16S ribosomal RNA (rRNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome [Shine and Dalgarno, Nature 254: 34-38 (1975)].
Translation initiation signals may vary in "strength" as measured by the quantity of protein synthesized relative to the amount of gene-specific mRNA. Any SD-AUG combination that can be utilized by host cell ribosomes may be employed; such combinations include, but are not limited to, the SD-AUG combination from the cro gene or the N gene of coliphage .lambda., or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-AUG combination produced by recombinant DNA or other synthetic technique may be used.
Any of the methods previously described (e.g., U.S. Pat. No. 4,237,224) for the insertion of DNA fragments into a vector may be used to ligate a promoter segment, a ribosome binding region and any other control elements into specific sites within the vector.
Similarly, a gene of interest (or any portion thereof) can be inserted into an expression vector at a specific site in relation to the promoter and other control elements so that the gene sequence can be expressed correctly on the plasmid. The resultant recombinant DNA molecule is then introduced into appropriate host cells by transformation, transduction or transfection (depending upon the vector/host cell system). Transformants are selected on the basis of the expression of an appropriate marker gene included, and known to be able to be expressed, on the vector in an appropriate host cell, such as ampicillin-resistance or tetracycline-resistance in E. coli, or thymidine kinase activity in eucaryotic host cell systems. Expression of such marker proteins indicates that the recombinant DNA molecule entered the cell and is intact. Expression vectors which are commercially available (Bethesda Research Laboratories, Inc., Rockville, MD) include the following vectors or their derivatives SV40 and adenovirus vectors, yeast vectors, bacteriophage vectors such as .lambda.gt-WES-.lambda.B, Charon 28, Charon 4A, gt-.lambda.BC, GT-1-.lambda.B, M13mp7, or plasmid DNA vectors such as pBR322, pAC105, pVH51, pACY177, pKH47, pACYC184, pUB110, pMB9, pBR325, ColE1, pSC101, pBR313, pML21, RSF2124, pCR1 or RP4.
In addition, host cell strains may be chosen which repress the action of the promoter unless specifically induced. Such host cells bear genes coding for repressor protein which specifically interacts with the operator region located near or overlapping the promoter to block transcription. In this way greater than 95% of the activity of the promoter can be repressed in uninduced cells. In certain operons, the addition of specific inducer substances (which prevent repressor protein from remaining bound to the operator) may be necessary for efficient transcription and translation of the inserted DNA. For example, the lac operon may be induced by the addition of lactose or IPTG (isopropylthio-.beta.-D-galactoside, an analog of lactose). A variety of other operons, such as trp, pro, etc., are under difference controls; the trp operon is derepressed when tryptophan is absent in the growth medium. The P.sub.R and P.sub.L promoters of .lambda. are derepressed by an increase in temperature if the temperature-sensitive repressor (product of the .lambda.cI857 gene) is used. Thus, expression of the inserted gene sequence can be controlled. This is important if the protein product of the cloned gene is lethal to host cells. In such cases, the foreign gene can be replicated but not expressed during growth of the transformants. After the cells reach a suitable density in the growth medium, the promoter can be induced for production of the protein.
Many factors complicate the expression of eucaryotic genes in procaryotes even after the proper signals are inserted and appropriately positioned. A clear understanding of the nature of these factors and the mechanisms by which they operate is presently lacking. One such factor is the presence of an active proteolytic system in E. coli and other bacteria. This protein-degrading system appears to selectively destroy "abnormal" or foreign proteins such as eucaryotic proteins. A tremendous utility, therefore, would be afforded by the development of a means to protect eucaryotic proteins expressed in bacteria from proteolytic degradation. One strategy is to construct hybrid genes in which the eucaryotic sequence is ligated in phase (i.e., in the correct reading frame) with a procaryotic gene resulting in a fusion protein product (a protein that is a hybrid of procaryotic and foreign or eucaryotic amino acid sequences) thereby perhaps causing the cell to regard the protein as "self" and spare it from proteolysis.
Construction of hybrid genes was the approach used in the molecular cloning of genes encoding a number of eucaryotic proteins, such as somatostatin [Itakura et al., Science 198: 1056 (1977)], rat pre-proinsulin [Villa-Komaroff et al., Proc. Natl. Acad. Sci., U.S.A. 75: 3727 (1978)], growth hormone [Seeburg et al., Nature 276: 795 (1978)], and ovalbumin-like protein [Mercereau-Puijalon et al., Nature 275: 505 (1978)]. Additionally, procaryotic promoters have been ligated to such fusion gene sequences in the case of ovalbumin [Fraser et al., Proc. Natl. Acad. Sci., U.S.A. 75: 5936 (1978)]and .beta.-globin [Guarente et al., Cell 20: 543 (1980)]. Although the molecular cloning and expression of several eucaryotic genes has been accomplished (see e.g., U.S. Pat. No. 4,332,892 to Ptashne et al. and U.S. Pat. No. 4,338,397 to Gilbert and Talmadge), the state of the art is such that expression of foreign or eucaryotic genes in procaryotic host cells can not be routinely performed.